In the last year we have purified the tissue protease inhibitor protease nexin (PN) and now are beginning to study its roles in regulating protease activity in the extracellular environment. We have shown that, in cultures of normal human fibroblasts, PN is secreted along with a proenzyme form of urokinase, a plasminogen activator (PA). When the proenzyme is activated, it is sensitive to inhibition by PN. Secretions of PN and the proenzyme are coordinately stimulated by growth factors or tumor promoter. We now will investigate whether the well-documented elevated release of active PA by malignant cells is caused by cellular release of active PAs, insufficient cellular release of PN, or caused by abnormal properties of their PN or PA. We will examine the ability of PN produced by normal cells to complex PAs released by malignant cells. We will establish how cellular release of PN by malignant cells is regulated when cellular expression of PA is modulated by glucocorticoids, growth factors and tumor promoters. In addition, we will begin to carry out a biochemical characterization of PN. The binding affinity of PN for a variety of different serine proteases will be determined in an effort to identify key physiological protease targets of this inhibitor.